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"Easiest" sticky end combination

ligation cloning restriction endonuclease

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3 replies to this topic

#1 doxorubicin

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Good

Posted 21 May 2013 - 03:45 AM

All else being equal, what two restriction enzymes when used together yield the insert that is easiest to insert (single copy) in the correct orientation?

#2 phage434

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Excellent

Posted 21 May 2013 - 06:27 AM

I like the Biobrick choice of EcoRI and PstI. Both are cheap, reliable, can be heat killed, work in the same buffer, are insensitive to E. coli methylation, and are available in high fidelity versions from NEB. They produce different overhangs: EcoRI a 5' overhang, and PstI a 3' overhang, so mis-ligation is unlikely. They also both cut near the end of dsDNA fragments with only a few bases outside the RE site.

#3 GNANA

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Neutral

Posted 21 May 2013 - 06:29 AM

I 'll go with the combination of enzymes that has higher cleavage efficiency (towards the end of oligos) in short time as per NEB like Bam HI, ECORI, Kpn and Xba (ofcourse there are some more), besides Hind III, Not1 are also easier....
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#4 notjustalabtech

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Posted 09 June 2013 - 11:46 AM

My favorites are EcoRI, BamHI, and Xba1





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